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#!/usr/bin/python
import sys, bisect, argparse, tarfile, StringIO, os, pwd
import grp, logging, time, copy
from tempfile import NamedTemporaryFile
from collections import Counter, defaultdict
from string import maketrans
import pysam
from pbsuite.utils.setupLogging import setupLogging
USAGE="""\
Parse and cluster mapped tails from a bam to create breakpoint candidates."""
class Bread():
"""
Holds a read that has a break in it
and all relevant information for clustering
"""
def __init__(self, read, readRef, log='h'):
"""
extract information from pysam.AlignedRead
if log=='h' get higher quality end
if log=='p' get prolog only
if log=='e' get epilog only
"""
self.read = read
self.readRef = readRef
if read.is_reverse:
begin = read.aend
end = read.pos
strand = 1
else:
begin = read.pos
end = read.aend
strand = 0
foundBreak = False #for the p only and looking for e or viceversa
self.proref = getTag(read, "PR")
self.prostr = getTag(read, "PI")
self.propos = getTag(read, "PP")
self.promaq = getTag(read, "PQ")
self.prorem = getTag(read, "PS")
# if we have an pro and (we want hq or we want pro
if self.propos is not None and (log == 'h' or log == 'p'):
foundBreak = True
if self.propos <= begin:
s, e, a, b, uq, dq, uR, dR = (self.propos, begin, \
"p", "i", self.promaq, \
self.read.mapq, self.proref, \
readRef)
ud = '3' if self.prostr == 0 else '5'
dd = '5' if self.read.is_reverse else '3'
else:
s, e, a, b, uq, dq, uR, dR = (begin, self.propos, \
"i", "p", self.read.mapq, \
self.promaq, readRef, \
self.proref)
dd = '3' if self.prostr == 0 else '5'
ud = '5' if self.read.is_reverse else '3'
rmSeq = self.prorem if self.prorem is not None else 0
inv = False if self.prostr == strand else True
self.epiref = getTag(read, "ER")
self.epistr = getTag(read, "EI")
self.epipos = getTag(read, "EP")
self.epimaq = getTag(read, "EQ")
self.epirem = getTag(read, "ES")
#Choose higher quality or force epilog
# if we have an epi and ((we want hq and it's of higher quality) or we want epi
if (self.epipos is not None) and ((log == 'h' and self.epimaq > self.promaq) or log == 'e'):
foundBreak = True
if self.epipos <= end:
s, e, a, b, uq, dq, uR, dR = (self.epipos, end, \
"e", "i", self.epimaq, \
self.read.mapq, self.epiref, \
readRef)
ud = '3' if self.epistr == 0 else '5'
dd = '5' if self.read.is_reverse else '3'
else:
s, e, a, b, uq, dq, uR, dR = (end, self.epipos, \
"i", "e", self.read.mapq, \
self.epimaq, readRef, \
self.epiref)
dd = '3' if self.epistr == 0 else '5'
ud = '5' if self.read.is_reverse else '3'
rmSeq = self.epirem if self.epirem is not None else 0
inv = False if self.epistr == strand else True
self.has_tail = foundBreak
if not self.has_tail:
return
self.uRef = uR
self.dRef = dR
#reference key; for sorting
j = [uR, dR]; j.sort()
self.refKey = "_".join(j)
#Points
self.uBreak = s
self.dBreak = e
#P,I,E
self.uTail = a
self.dTail = b
#Strands
self.uDir = ud
self.dDir = dd
#Mapqs
self.uMapq = uq if uq is not None else 255
self.dMapq = dq if dq is not None else 255
#Inv
self.isInverted = inv
#Remain
self.remainSeq = rmSeq
def near(self, other):
"""
Is this Bread and it's mate near the other Bread
"""
#Same target
if self.refKey != other.refKey:
return False
# are our components within buffer bp of each other
if abs(self.uBreak - other.uBreak) > BUFFER:
return False
if abs(self.dBreak - other.dBreak) > BUFFER:
return False
"""
del
->p| |i-> ud=3,dd=3
->i| |e-> ud=3,dd=3
<-i| |p<- ud=5,dd=5
<-e| |i<- ud=5,dd=5
ins gain same as del but close can estimate size from remaining
ins sequence
|i<- <-e| ud=5,dd=5
|p<- <-i| ud=5,dd=5
|e-> ->i| ud=3,dd=3
|i-> ->p| ud=3,dd=3
inv sequence
->p| <-i| ud=3,dd=5
->i| <-e| ud=3,dd=5
|i-> |p<- ud=3,dd=5
|e-> |i<- ud=3,dd=5
|p<- |i-> ud=5,dd=3
|i<- |e-> ud=5,dd=3
<-i| ->p| ud=5,dd=3
<-e| ->i| ud=5,dd=3
missed adapter evidence is any one of these. (a || b)
both would suggest some kind of duplication inversion
but that requires non-local information.
These are the same as the inversion/but the sequence is
on top of itself
->i| a ud=3
<-e| a dd=5
->p| a ud=3
<-i| a dd=5
|e-> b ud=3
|i<- b dd=5
|i-> b ud=3
|p<- b dd=5
"""
#brute force
#ins = ["i<-=<-e", "p<-=<-i", "e->=->i", "i->=->p"]
#dele = ["->p=i->", "->i=e->", "<-i=<-p", "<-e=i<-"]
#inv = ["->p%<-i", "->i%<-e", "i->%p<-", "e->%i<-", \
#"p<-%i->", "i<-%e->", "<-i%->p", "<-e%->i"]
#tloc = ["->i=e->", "->p=i->", "<-i=p<-", "<-e=i<-",\
#"i->=->p", "i->=->p", "p<-=i<-", "i<-=e<-"]
#if self.bpStr() in ins and other.bpStr() in ins:
#return True
#if self.bpStr() in dele and other.bpStr() in dele:
#return True
#if self.bpStr() in inv and other.bpStr() in inv:
#return True
#if self.uRef != self.dRef
#return False
# are we moving in the same direction
# this creates 2 cluters - one per strand
if self.annotate() != other.annotate():
return False
if self.uDir == other.uDir and self.dDir == other.dDir:
return True
elif self.read.is_reverse != other.read.is_reverse:
#If we're on opposite strands,
#but our pieces are pointing together
if self.uDir != other.uDir and self.dDir != other.dDir:
return True
else:
if self.uTail == 'i' and other.uTail in ['p', 'e'] or \
self.dTail == 'i' and other.dTail in ['p', 'e']:
#This can't be true
if self.uDir == other.dDir and self.dDir == other.uDir \
and self.read.is_reverse == other.read.is_reverse:
return True
return False
def getInvStr(self):
return "%" if self.isInverted else "="
def getRevStr(self):
return "<-" if self.read.is_reverse else "->"
def annotate(self):
"""
based on the properties of orientation, create annotation
of what possible variant is here
"""
ins = ["i<-=<-e", "p<-=<-i", "e->=->i", "i->=->p"]
dele = ["->p=i->", "->i=e->", "<-i=p<-", "<-e=i<-"]
inv = ["->p%<-i", "->i%<-e", "i->%p<-", "e->%i<-", \
"p<-%i->", "i<-%e->", "<-i%->p", "<-e%->i"]
if self.uRef != self.dRef:
self.estsize = -1
return "TLOC"
bps = self.bpStr()
if bps in ins:
if abs(self.uBreak - self.dBreak) < 100:
#rmSeq = self.epirem if self.epirem is not None else 0
self.estsize = self.remainSeq
else:
self.estsize = abs(self.uBreak - self.dBreak)
return "INS"
if bps in dele:
self.estsize = abs(self.uBreak - self.dBreak)
return "DEL"
if bps in inv:
self.estsize = abs(self.uBreak - self.dBreak)
return "INV"
#never gets here... unless XinvxX
return "UNK"
if self.uRef == self.dRef:
if self.uDir != self.dDir:
self.estsize = abs(self.uBreak - self.dBreak)
return "INV"
if self.dBreak - self.uBreak < 100 and self.remainSeq >= 100:
self.estsize = self.remainSeq
return "INS"
elif self.uDir == self.dDir:
ut, dt = self.__tailtoint__()
self.estsize = abs(self.uBreak - self.dBreak)
if (self.uDir == '3' and ut > dt) or (self.dDir == '5' and ut < dt):
return "INS"
else:
return "DEL"
else:
self.estsize = -1
return "TLOC"
def __tailtoint__(self):
"""
returns the uTail and dTail as ints
"""
trans = {"p":1,
"i":2,
"e":3}
x = trans[self.uTail]
y = trans[self.dTail]
return x, y
def bpStr(self):
def swap(a, b):
trans = maketrans("<>","><")
if a in ['p','i','e']:
b = b.translate(trans)[::-1]
elif b in ['p','i','e']:
a = a.translate(trans)[::-1]
return (b, a)
x, y = self.__tailtoint__()
if (x < y):
uC = ("->", self.uTail)
dC = (self.dTail, "->")
elif (x > y):
uC = (self.uTail, "->")
dC = ("->", self.dTail)
if self.uDir == '5':
uC = swap(*uC)
if self.dDir == '5':
dC = swap(*dC)
return "".join(uC) + self.getInvStr() + "".join(dC)
def anyNone(self):
"""
This is an old debugging method
"""
if self.uTail is None:
logging.debug("uTail none %s" % str(self.read.qname))
elif self.uMapq is None:
logging.debug("uMapq none %s" % str(self.read.qname))
elif self.uDir is None:
logging.debug("uDir none %s" % str(self.read.qname))
elif self.uBreak is None:
logging.debug("uBrea none %s" % str(self.read.qname))
elif self.getInvStr() is None:
logging.debug("getIn none %s" % str(self.read.qname))
elif self.getRevStr() is None:
logging.debug("getRe none %s" % str(self.read.qname))
elif self.dBreak is None:
logging.debug("dBrea none %s" % str(self.read.qname))
elif self.dDir is None:
logging.debug("dDir none %s" % str(self.read.qname))
elif self.dMapq is None:
logging.debug("dMapq none %s" % str(self.read.qname))
elif self.dTail is None:
logging.debug("dTail none %s" % str(self.read.qname))
def getBriefData(self):
"""
gets the relevant data in a list
"""
return [self.uRef, self.uBreak, self.uMapq, \
self.dRef, self.dBreak, self.dMapq, \
self.remainSeq, self.read.qname]
def __str__(self):
"""
Need to update this...
Outputs the string representation of the bread
chrom uTails uMapq uDirs uBreak isInv dBreak dDirs dMapq dTails remainSeq
chrom_chrom uRef uTail uMapq uDir uBreak isInv remainSeq dBreak dDir dMapq dTail dRef
#id chrKey
uRef uBreak uMapq dRef dBreak dMapq remainSeq numReads numZMWs evidence
"""
data = self.getBriefData()
data.insert(-1, self.bpStr())
data.insert(-1, self.annotate())
# ur ub uq dr db dq rs bp an rn
return "%s %d %d %s %d %d %d %s %s %s" % tuple(data)
def __lt__(self, other):
return self.uBreak < other.uBreak
def __gt__(self, other):
return self.uBreak > other.uBreak
class Bnode(Bread):
"""
A node for a sorted list where a cluster of similar breakpoint
are pooled
"""
def __init__(self, bread):
"""
start with a point
"""
Bread.__init__(self, bread.read, bread.readRef)
self.breads = [bread]
def estimateEnds(self):
"""
Go through all of the breaksand try to make the best estimate
of where they are.
I'm going to create a mode average median of the breaks
"""
upstream = map(lambda x: x.uBreak, self.breads)
self.upPos = Counter(upstream).most_common()
self.avgUpPos = sum(upstream)/len(upstream)
dnstream = map(lambda x: x.dBreak, self.breads)
self.dnPos = Counter(dnstream).most_common()
self.avgDnPos = sum(dnstream)/len(dnstream)
def breadMatch(self, bread):
"""
Checks if the bread is near any of the bread in this node
"""
for i in self.breads:
if bread.near(i):
return True
return False
def addBread(self, bread):
"""
put more bread in the basket
"""
self.breads.append(bread)
def numReads(self):
"""
returns the number of reads supporting in the node
"""
return len(self.breads)
def numUniqueReads(self):
"""
returns the number of unique reads in the node
"""
count = Counter([x.read.qname for x in self.breads])
return len(count)
def numUniqueZMWs(self):
x = []
for y in self.breads:
x.append('/'.join(y.read.qname.split('/')[:2]))
return len(Counter(x))
def avgMapq(self, threshold=None):
"""
return average mapping quality
"""
if threshold is None:
x = []
for y in self.breads:
x.append(y.uMapq); x.append(y.dMapq)
return sum(x)/float(len(x))
else:
us = []
ds = []
for y in self.breads:
us.append(y.uMapq)
ds.append(y.dMapq)
us = sum(us)/float(len(us))
ds = sum(ds)/float(len(ds))
return (us >= threshold) and (ds >= threshold)
def avgRemainSeq(self):
"""
return the average length of the remaining sequence
"""
x = 0
for y in self.breads:
x += y.remainSeq
return x/len(self.breads)
def toPrettyStr(self):
"""
Gets the average positions and mapqs, places that into
the parent Bread, and returns a fully formatted string
"""
self.estimateEnds()
self.uBreak = self.avgUpPos
self.dBreak = self.avgDnPos
#This should be in a method...
self.uMapq = sum(map(lambda x: x.uMapq, self.breads)) / len(self.breads)
self.dMapq = sum(map(lambda x: x.dMapq, self.breads)) / len(self.breads)
readData = set() #getBPstr
for i in self.breads:
readData.add(i.bpStr())
readData = list(readData)
readData.sort()
self.remainSeq = self.avgRemainSeq()
data = Bread.getBriefData(self)[:-1]
data.append(self.annotateBnode())
data.append(self.numUniqueReads())
data.append(self.numUniqueZMWs())
data.append(";".join(readData))
return "\t".join([str(x) for x in data])
def toBriefString(self):
"""
"""
self.estimateEnds()
anno = self.annotate()
return "{uRef}{bps}{dRef}:{start}-{end}({svtype}){size}*{count}".format(\
uRef=self.uRef, bps=self.bpStr(), dRef=self.dRef, \
start=self.avgUpPos, end=self.avgDnPos, svtype=anno, \
size=self.estsize, count=len(self.breads))
def annotateBnode(self):
"""
Does the potentially ambiguous annotation
"""
x = Counter([x.annotate() for x in self.breads])
anno = x.most_common()[0][0]
if len(x) != 1:
anno += '*'
return anno
def __str__(self):
ret = "Bnode w/ %d Breads %d unique sub %d unique zmws\n" % \
(len(self.breads), self.numUniqueReads(), self.numUniqueZMWs())
for i in self.breads:
ret += str(i)+"\n"
return ret
def makeBreakReads(bam, minMapq=150, buffer=500, getrname=None):
"""
Extracts all of the tail-mapped reads from a bam and crates break reads (Bread)
that are then bisect placed inside of
getrname is bam.getrname method. I have this because sometimes we call tails within
a bam.fetch region and I want to be able to call makebreakreads on that, which returns
an iterator -- If you don't know what I'm talking about, leave getrname blank
I should iter on a per chromosome basis
"""
if getrname is None:
getrname = bam.getrname
tlocs = defaultdict(list)
for chrom, length in zip(bam.references, bam.lengths):
logging.info("Parsing %s" % chrom)
ret, t = parseBreakReads(bam.fetch(reference=chrom, start=0, \
end=length), \
getrname, minMapq)
for key in t:
tlocs[key].extend(t[key])
if len(ret.keys()) != 0:
yield ret
del(ret)
for refKey in tlocs:
logging.info("Parsing %s" % refKey)
ret, x = parseBreakReads(tlocs[refKey], getrname, minMapq, True)
logging.debug(ret)
if refKey in ret.keys():
yield {refKey:ret[refKey]};#len(ret.keys()) == 0:
del(ret)
def parseBreakReads(reads, getrname, minMapq=150, isTloc=False):
"""
Need to separate parsing an entire bam and parsing a set of reads so
that I can re-enable ForceCalling
"""
#need to call a method that takes a list here.. returns
ret = {}
#Tlocs are still going to be fucked
tlocs = defaultdict(list)
for read in reads:
refName = getrname(read.tid)
#skip non-primaries
if not (read.flag & 0x1) or (read.flag & 0x40 or read.flag & 0x80):
continue;
#just Put P and E in separate Breads - ensure they both exist
panP = Bread(read, refName, log="p")
panE = Bread(read, refName, log="e")
pans = []
if panP.has_tail:
pans.append(panP)
if panE.has_tail:
pans.append(panE)
###### Need to do this twice if P and E
for pan in pans:
refKey = pan.refKey
if len(set(refKey.split('_'))) != 1 and not isTloc:
tlocs[refKey].append(read) #copy.copy(read))
continue #I'm breaking the tlocs for now
#of quality
if pan.uMapq < minMapq or pan.dMapq < minMapq:
logging.debug("read %s mapq is too low (uMapq %d - dMapq %d)" % (read.qname, pan.uMapq, pan.dMapq))
continue
if refKey not in ret.keys():
ret[refKey] = []
clist = ret[refKey]
#point = bisect.bisect_left(clist, pan)
point = bisect.bisect(clist, pan)
unear = False; dnear = False
#while moving upstream and I'm within buffer,
#see if I've got someone to merge with
lpoint = point
while lpoint > 0 and abs(pan.uBreak - clist[lpoint - 1].uBreak) <= BUFFER:
if clist[lpoint-1].breadMatch(pan):
unear = True
break
lpoint -= 1
dpoint = point
while dpoint < len(clist) and abs(pan.uBreak - clist[dpoint].uBreak) <= BUFFER:
if clist[dpoint].breadMatch(pan):
dnear = True
break
dpoint += 1
if not (unear or dnear):
bisect.insort( clist, Bnode(pan) )
elif unear and not dnear:
clist[lpoint-1].addBread( pan )
elif not unear and dnear:
clist[dpoint].addBread(pan)
elif unear and dnear:
node = Bnode( pan )
for i in clist[lpoint-1].breads:
node.addBread( i )
for i in clist[dpoint].breads:
node.addBread( i )
del(clist[dpoint])
del(clist[lpoint-1])
bisect.insort( clist, node )
return ret, tlocs
#return tloc
def bNodeMerge(node1, node2):
ret = Bnode(node1.read)
def getTag(read, tagId):
"""
Returns the specified tag or None from an AlignmentRecord
"""
for i in read.tags:
if i[0] == tagId:
return i[1]
return None
def parseArgs(argv):
parser = argparse.ArgumentParser(prog="Honey.py tails", description=USAGE, \
formatter_class=argparse.RawDescriptionHelpFormatter)
parser.add_argument("bam", metavar="BAM", type=str, \
help="BAM containing mapped reads")
parser.add_argument("-B", "--buffer", type=int, default=1000, \
help=("Buffer around breaks reads must fall "
"within to become clustered (%(default)s)"))
parser.add_argument("-b", "--minBreads", type=int, default=3,\
help="Minimum number of reads (%(default)s)")
parser.add_argument("-z", "--minZMWs", type=int, default=3, \
help="Minimum number of unique ZMWs (%(default)s)")
parser.add_argument("-q", "--minMapq", type=int, default=150, \
help="Minimum mapping quality of a read and it's tail to consider (%(default)s)")
parser.add_argument("-f", "--fastq", action="store_true", \
help="Write fastq for each cluster into a .tgz archive (%(default)s)")
parser.add_argument("-o", "--output", type=str, default=None, \
help="Output file to write results (BAM.hon.tails)")
# parser.add_argument("--noAdaptFilter", action="store_false", \
# help="Keep reads that appear to have a missed adapter orientation")
#parser.add_argument("-a", "--ambigous", action="store_true",
#help="Report SVs with ambigous annotation e.g. INS* (False)")
parser.add_argument("--debug", action="store_true")
parser.add_argument("-v", "--verboseFile", action="store_true", \
help="Print each read inside of a cluster to <output>.verbose (%(default)s)")
args = parser.parse_args(argv)
global BUFFER
BUFFER = args.buffer
if args.output is None:
args.output = args.bam[:-4] + ".hon.tails"
if args.verboseFile:
args.verboseFile = args.output + '.verbose'
setupLogging(args.debug)
return args
def run(argv):
args = parseArgs(argv)
bam = pysam.Samfile(args.bam,'rb')
try:
if bam.header["HD"]["SO"] != "coordinate":
logging.warning("BAM isn't marked as sorted. Results may be wrong if this is correct.")
except KeyError:
logging.warning("Assuming BAM is sorted by coordinate. Results may be wrong if this is incorrect.")
if args.fastq:
tarOut = tarfile.open(args.output+".tgz", 'w:gz')
fout = open(args.output,'w')
fout.write("#Args: %s\n" % str(args))
#uChrom dChrom
fout.write(("#id\tchrKey\tuRef\tuBreak\tuMapq\tdRef\tdBreak\tdMapq"
"\tremainSeq\tannot\tnumReads\tnumZMWs\tevidence\n"))
if args.verboseFile:
vOut = open(args.verboseFile, 'w')
vOut.write("#uRef uBreak uMapq dRef dBreak dMapq remainSeq break annot readName\n")
clu = 0
for retDict in makeBreakReads(bam, minMapq=args.minMapq):
points = retDict
chrom = retDict.keys()[0]
#print "chrom", i, "-", len(points[i]),"clusters"
logging.info("Chrom %s made %d pre-filter clusters" % (chrom, len(points[chrom])))
postCnt = 0
for j in points[chrom]:
#filtering from parameters
if j.numUniqueReads() < args.minBreads or \
j.numUniqueZMWs() < args.minZMWs:
continue
postCnt += 1
fout.write(str(clu) + "\t" + chrom + "\t" + j.toPrettyStr()+"\n")
if args.fastq:
fastq = StringIO.StringIO()
tfn = NamedTemporaryFile(suffix=".bam", delete=False).name
align = pysam.Samfile(tfn, 'wb', template=bam)
for r in j.breads:
read = r.read
fastq.write("@%s\n%s\n+\n%s\n" % (read.qname, read.seq, read.qual))
align.write(read)
info = tarOut.tarinfo()
info.name = "clu%d.fastq" % (clu)
info.uname = pwd.getpwuid(os.getuid())[0]
info.gname = grp.getgrgid(os.getgid())[0]
info.size = fastq.len
info.mtime = time.time()
#print dir(info)
fastq.seek(0)
align.close()
tarOut.addfile(info, fastq)
tarOut.add(tfn, arcname="clu%d.bam" % clu)
os.remove(tfn)
if args.verboseFile:
vOut.write("##Cluster %d - %s\n" % (clu, chrom))
for r in j.breads:
vOut.write(str(r)+'\n')
fout.flush()
clu += 1
logging.info("Chrom %s made %d post-filter clusters" % (chrom, postCnt))
fout.close()
if args.fastq:
tarOut.close()
if args.verboseFile:
vOut.close()
logging.info("Finished")
if __name__ == '__main__':
run(sys.argv[1:])
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